The likelihood of transmitter release from presynaptic release sites is regulated by several factors including the frequency of action potentials and the activity of presynaptic receptors. In addition to these mechanisms, subthreshold depolarizations of the somatodendritic compartment recently have been shown to alter action potential-driven release at distant locations of the axon. This form of regulation of release is analog in nature, i.e., though it alters the probability of release to an action potential, its potential to alter reease requires neither action potentials nor local receptor activation. Rather, these subthreshold depolarizations passive spread through the axon and are reported to affect release by calcium-dependent and calcium-independent mechanisms. Analog signaling adds another dimension to the control of neuronal circuit function which depends not only on the history of action potential frequency but also on the subthreshold changes in membrane potential that preceded a given action potential. Presynaptic receptors can also regulate release probability and alter the axonal membrane potential. The changes in axonal membrane potential can passively propagate antidromically and alter the excitability of the axon initial segment, a function which until recently was thought to be the exclusive domain of synapses on the somatodendritic membrane. The objective of this proposal is to determine the mechanisms underlying orthodromic and antidromic analog signaling and to determine if these mechanisms are used generally in the CNS. Orthodromic signaling will be studied in three dissimilar neurons, cerebellar molecular layer interneurons, dentate gyrus granule cells and cortical layer 5 pyramidal cells. The proposed mechanisms for orthodromic signaling in these three cells types are contradictory but include both calcium-dependent and calcium-independent processes. Antidromic signaling has only been demonstrated in one neuronal type, cerebellar granule cells. Though a number of axonal receptor types may affect initial segment excitability, NMDA receptors are particularly interesting candidates because of their use-dependence. Abundant evidence indicates that cortical layer 4 spiny stellate neurons in both visual and barrel cortex express presynaptic NMDA receptors that alter release properties and are required for the induction of long term depression. We will use two photon laser scanning microscopy, two photon laser uncaging and electrophysiology to determine the mechanisms of orthodromic analog signaling and the extent of presynaptic NMDA receptor expression, the physiological conditions necessary for their activation and their effects on the excitability of the axon initial segments of the layer 4 spiny stellate neurons. PUBLIC HEALTH RELEVANCE: Understanding how the brain functions, and what goes awry in disease states, depends on knowledge of the fundamental unit of information processing within the brain called the synapse. To understand the synapse, we need detailed information about its physical structure, molecular constituents, biochemical and physiological mechanisms and how these properties change with age and experience. Rational design of therapies for neurological deficits is not possible without a fundamental understanding of synaptic function.